CA2376199A1 - Use of verapamil and verapamil derivatives for producing medicaments with an inhibiting effect on .beta.-glucuronidase in human tissue - Google Patents
Use of verapamil and verapamil derivatives for producing medicaments with an inhibiting effect on .beta.-glucuronidase in human tissue Download PDFInfo
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- CA2376199A1 CA2376199A1 CA002376199A CA2376199A CA2376199A1 CA 2376199 A1 CA2376199 A1 CA 2376199A1 CA 002376199 A CA002376199 A CA 002376199A CA 2376199 A CA2376199 A CA 2376199A CA 2376199 A1 CA2376199 A1 CA 2376199A1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/275—Nitriles; Isonitriles
- A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/275—Nitriles; Isonitriles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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Abstract
The invention relates to the use of verapamil or verapamil derivatives for producing medicaments which have an inhibiting effect on .beta.-glucuronidase in human tissue.
Description
' ' CA 02376199 2001-12-04 Use of veravamil and verapamil derivatives for the preparation of pharmaceuticals with s-~lucuronidase inhibitin~ action in human tissue The subject of the present invention is the use of verapamil or verapamil~derivatives in pharmaceuticals for the inhibition of the enzyme beta-glucuronidase in human tissue with the object directly to achieve therapeutic effects or to improve its therapeutic breadth by combined use together with glucuronidated or glucuronidatable active materials, The conjugation of endogenic or exogenic substances with glucuronic acid is an important metabolic reaction in humans~and animals. Glucuronic acid can be conjugated with the most varied substances, e,g, pharmaceutically active materials and their metabolites, The conjugst~on reaction takes place by transfer of activated glucuronic - said (UDP-glucuronic acid) to the substrate by means of the enzyme glucuronal transferase. In general, the organism uses the conjuga Lion- reaction for ~ietoxication since glucuronic acid conjugates are usually less toxic and, on the basis of their good water solubility, are easily excreted via the kidneys~o~rthe gall secretions via the intestsnes, A conjugatibv ~ can also take place in non-enzymatic wags by chemical synthesis.
The glucuronic acid conjugates can, howeverv, also be cleaved by catalytic action of glucuronidases:into glucuronic acid and in to the starting product,. The cleavage of glucuronides frequently takes place after excretion thereof via the bile in deeper lying small intestine sections or in the large intestine, The thereby resulting starting substances can again be resorbed~snd this become renewed a cttEve in the organism. This process, designated as enterohepatic circulation, can prolong the desired action of substances but can also increase the toxic actions of poisonous substances.
' CA 02376199 2001-12-04 By medicamentous regulation of the beta-glucuronidase activity in the various tissues, new therapy concepts are opened up,.
Use of glucuronidase inhibitors its cancer therapy, A peculiarity of cancer tissues is their high con-centration of bets-glucuronidaees or an extremely high glucuronidase activity, Closely associated with the increased glucuronidase activity is the tendency to form certain tumour meta~~tases, By general administration of a beta-glucuronidase inhibitor, in the case of tumours which, on the basis of the increased beta-glucuronidase ac-tivity, tend to the progression and metastasis formation, the tumour spreading out is seduced via the inhibition of the tumour glucuronidase, Saccharo-1,4-lactose, 2-acetamidoglycal and heparin derivatives were tested for :this purpose /Bernacki R,J " Cancer Metastasis Rev,, (1985) 4: 81 - lOl;Nakajima M,, Journal of Cellular Biochemistry (1988) 36: 15~ - 16'7; Niwa T,, Journal of Biochemistry (1972 ) '72: 20'7 - 211, In most recent times, selective glucuronidase inhibitors have been s9nthe~d (Bosslet K~, EP 0822192), Besides the general use for the therapy, glucuronid~rse .
inhibitors can also be used supportingly in the chemo-therapy of cancer patients f or the increasing of the desired effect in the case of simultaneous reduction of the undesired actions,.
The chemotherapy causes an extraordinary physical and psychic stressing of the cancer patient, Giucuronidase inhibitors can ameliorate negative actions of the chemo-therapy and simultaneously increase the effectiveness of the therapy, For this purpose, the following starting points present themselves, Chemotherapeutics are, inter alia, also excreted via their glucuronides via small intestines, Due to the actions of the there-present glucuronidases, there takes place a cleavage of these glucuronides and liberation ~
of the active cell-toxic substances which damage the intestinal tissue present in continuous cell division and regeneration, For the patient, there result therefrom nausea, vomiting and diarrhoea, combined with a fluid and weight loss, Beta-glucuronidase inhibitors can protect the intest-ines against toxic products from cytostatic gluc~uronides, Thus,e,g, the intestinal toxicity of the anti-tumour agent irinote.ctan hydrochloride can be minimised by preventative 1!Q administration of the beta-glucuronidase inhibitor baicalin, The patients arethe~s protected against a massive diarrhoea and the fhuid losses involved therewith L~akasuna K, Jpn,.
Cancer Res, (1995) 86:: 9'78 - 84;: Kemataki T, HB~, Pat, 5,44,719).
Consideraaions exist of using the cleavage of glucur-onide in certain tissues in order to liberate the active substances from inactive: precursors of a~:ctive medicament (prodrugs), Due to the preferred. liberation in the dises ed target tissues, via the inc:ressed substance concentration, there can be achieved a more or less local action in the case of low ~r~stemic actia~n ~~p~erker B,.,, Clin,. Phsrmakinet, (1_997) 33:: 18 - 31~,. This therapy possibility would be of interest above all in the case of the use of s~.de effect-rich substances in tumour therapy 5 bs9cause tHe. d.e irked aytaaogic properties of chemothera-peutics can be concentrated on the tumour tissues" The tumour progression and the me sstasis formaaion is frequently bound up with an inc:rea ~ed gl.ucuronida~se activity,. In necrotic tumour regions, sn ~~ncraa:aed ~0 glucuronidase activity is present in the extrscel7~uTar space wherees in the healthy tissue the glucuronida a activity is substantially intracellular localised, A
pH value in the tumour displaced towards acid can again increase the activity of the beta-glucuronidase, These 35 physiological conditions offer starting points for the application of glucuronic acid conjugates with c hemotherapeutics to tumour patients for the local ' ~ CA 02376199 2001-12-04 .~5_ liberation of the active substrate after cleavage b9 the:
locally increased glucuronidase activity LSperrker B,.,.
Clin, Pharmacokinet, (199?) 33:~ 18 - 317, The local action could be strengthenedby simultaneous administration of a glucuronide prodrug and of, s tumour-specific antibody which is covalently bound with beta-glucuronidase (antibody-directed prodrug therapy = ADEPT)LSperker B,, CZin, Pharmacokinet, (1997) . 33:~ 18 - 317, The increased tumour selectivity of glucuronide prodrugs leads iro correspondingly higher active material levels in the tumour and sumultaneously to lower native material concentrations in healthy tissue regions, i,e, the effectivenesse~ and compatabilities of the chemo-therapeutics are increased, Known examples are doxorubicin glucuronide prodrugs which, in comparison with the free doxorubicin, make .possible in tumour tissues an about 10 times higher doxorubicin level but, at the same time, protects healthy tissue with a lower concentration so that e,g, the typical cardiotoxic property ~f doxorubicin only plays a subsidiary role L$osslet K,, Cell Biophys, (1994) 24-25;:
51-63; Bosslet K,, Cancer Res, (1994) ~.: 2151-g;
Bosslet K,, Cancer Res, (1998): 1195 - 201; Murdter, T,E "
Cancer Res, (199'7) 5~: 2440-~~, None of these investigations has hitherto lead to therapeutically usable results, i,e, utilisable medicaments, Description of the invention The invention has set itself the task of finding glucuronidase inhibitors which are otherwise pharma-cologically not or only little effective, i,e, display few side reactions, in order to use these as medicaments in the above-described uses alone or in combination with other medicaments for the increasing of the therapeutic breadth, New pales 6 - 11 filed 15th December, 2000 In the case of administration of biologically-inactive glucuronide prodcugs, together with a beta-glucuronidase inhibitor, the cleavage into the effective substrate is delayed so that, in the case of prodrugs with long elimination half value time, the systemic availability - is prolonged,. Correspondingly, the dose can be reduced end the dose ging interval lengthened, Im the case of the tumour-specific prodaug therapy, by additional administration of a cell membrane-premeable IO beta-glucuronidase inhibitor, such as verapamil, the therapeutic breadth is thereby increased that the sub-star~t~.slly intracellularly present beta-glucuronida se is inhibited in healthy tissue and a pharmacological action is~thereby hindered, In the tumour tissue, due to the physiological or due to the glucuronidase concentration increased by ADEPT therapy, the effective substrate is, as previously, formed in the case of suitable choice of dose,.
The inhibiting action on the beta-glucuronidase activity claimed in the invention is verified in the results set out in the following,.
Iimestigations of the lowering of human ~-glucuronidase activity by verapamil, its metabolites and gallopamilr The calcium antagonist verapamil (not only racemate but also both enantiomers), its metabolites and the derivative gallopamil aae in the position Via' Lower the activity of the human ~-glucuronidases, A direct inhibition of the ~-glucuronidase sctivity could be shown in experiments with human ILiver homogenates, For this purpose, homogenates of various liver samples were: incubated with 2,.5 mM 4-methyl-belliferyl-~-D.t.
glucuronide (MUG) and analysed by means of Hi'~C, The:
concentrations of the liberated 4-methylumbelliferone is a measure of the activity of the ~-glucaronidase, In the case of homo mates which, in addition to MUG, also received T00 ~-M verapamil ( racemat ~), the activity was reduced significantly by about 25~, in comps~ison with AMENDED
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the control samples (Fig, 1).
Parallel bring about verapamil, the metabolite nor-verapamil, D~02, D~03 and gallopamil in the human hepatoma cell Mine HepG2 after 48 h incubation a reduction of the ~-glucuronidase activity to 50 - 65~ which is to be attributed to a reduced expression of the enzyme,.
This reduction of the activity in concentration dependent (Fig. 2).
The reduction of the ~-glucuronidase a3ctivity carz~ld be observed eqw-311y strongly with verapamil racemate and with R- and S-verapamil, fihe metabolites no~verapamil, D'702 and D703 show a comparable influence on the activity of the ~-glucuronidase in HepG2 cells, The incubation with D61~, a further metabolitew, only brings about a lowering of the activity by' 12~ whicli, however, ~s not statistically significant, Gallopamil brings about an effect comparable to verapamil (Fig" 3), -Example 1 Inhibition of the activity of human liver ~-glucuronidase by verapamil (F.2g, 1), Human liver homogenates were incubated with the enzyme substrate 4-methylbelliferyl-~-D-glucuronide (1 h, 3'7°C), 100 ~.M verapamil or DMSO (control) were added to the reaction mixture, The liberation of 4-methylumbelli-ferone was measured by mearns of HPI~C analysis ('~ significant difference to the control; p ~ 0001; n =
3 independent experiments), Exa mp 1 a 2 Concentration dependency of the verapamil action in the human hepatoma cell Mine HepG2 (Fig, 2), HepG2 cells were incubated for 48 h at 3'7oC with the concentrations of verapamil given in Fig, 2, A~'ter ~.~rsie of the cells, in each case 2,25 ~.g of cellular protein were incubated (2 h, 3'7°C) with ~-glucuronidase substrate 4-methylumbelliferyl-~-D-glucuronide and the concentration of the liberated 4-methylumbelliferone measured by means of HPI~C ( '* significant difference ~o the control, p <0~05), HMENDED
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Example 3 Lowering of the ~-glucuronidase activity in HepG2 cells by incubation with verapamil, verapsmil metabolites sand gallopamil (Fig, 3), HepG2 cells were incubated for 48 h,~t 37°C with 100 ~M verapamil (Yera), in each ceae 100 rM DFl'7, D~02, D'703, 30 ~,.M norverapamil (Nor) or 100 ~M gallopamil (Gallo), After lysis of the cells, the ~-glucuronidaee activity was determined by means of 4-methylumbelliferyl-~i-D-glucuronide cleavage (significant difference to the control, '~ P < 0,01, *~ g ~ 0,.001, n = 3 independent -experiments), Example 4 Lowering of the beta-glucuronidase expression by vera~amil - 15 in the human hepatoma cell line HepG2 (Fig,. 4), HepG2 cells were- incubsrted 48 h at 3~°C with 100 ~M
verapamiT or DMSO (control), After lysis of the cells, 50 ~g cellular protein were separated off by means of SDS page,. transferred to nitrocellulose and subsequently incubated with the monoclonal antibody 2156/42, The band intensity was determined densitometrically (DE = densito-metric units; 'significant difference to the control, p ~ 0,05; n = 3 independent experiments), Inhibition of the glucuronidases in the rat intestine by verapamil (comparison) In a study with Sprague-Dawley rats, the absorption of orally administered morphine-6-glucuronide (M6G) to twa groups .~. - (group- 1~. 5, without verapamil administ-ration; group 2: n = 4 previous verapamil afministration) was investigated, The study was carried out with rats since these cannot form M6G metabolically from morphine (Aasmundstad T,A,, Biochem, P.harmacol, (1993) 46:
961-968) so that the M6G measured in the plasma origi.na.r~ed= exclusively from the absorption of the orally administered M6G, Whereas the previous administration of verspamil had no influence on the height of the plasma concentration AMENDED
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of M6G or its variation in time, the concentrations of morphine and M3G in the case of previous verapamil administration (group 2) were distinctly smaller than in the case of the group without verapamil (group 1) (Fig. 5).
The absent influence on the height of the plasma concentration of M6G or its variation in time makes it improbable that the reduction of the morphine and M3G
absorption depends upon an inhmbition of the intestinal mobility L~hah M~H" J, Pharm, Pharmacol, (198?) 39:
103?-1038; Krevsky B,, Dig,. Dis.. Sci, (1992) 37:
919-9247, It is known that M6G inhibits the intestinal motility with the same potency as morphine ~Schmidt N,, Eur, J, Pharmacol, (1994) 255 245-237, An increase of this inhibition by verapamil ,L~hah N~,.H,., J,. Pharm, Pharmacol,. (198?) 39, 1037-103_87 acts with all probability on M6G and morphine to the same extent, On t he other hand, only the plasma level of morphine Qr M3G but not of M6G were reduced, i,e, the cleavage of M6G available after oral administration to morphine is thus inhibited, Therefrom result lower morphine and, as a result, M3G
plasma levels since the greater part of the absorbed morphine i5 metabolised by glucurony~. transfera.ses to M3G, The carrying out of the experiments i~ described in Example 5, Example 5 Plasma concentration time progression of morphine-6-glucuronide (M6G), morphine and morphine-3-glucuronide (M3G) after oral administration to Sprague-Ds~wley rats of M6G with or without pervious oral administration of verapamil (Fig,. 5), The investzigation was carried out on 9 male S:prsgue-Dawley rats, The rats were divided into 2 groups:: group T
(5 animals, weight: 258 + 31.2 g) received only 62,5 mg/kg morphine-6-glucuronide (M6G) administered orally, Group,2 (4 animals, weight 2?2 +-8 g) received, 15 minutes AMENDED
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before M6G administration (62,5 mg/kg orally-, ?0 mg/kg verapamil orally administered, The groups dia not differ significantly from ane another with regard to their weight (t-test: t = -0,923, p = 0,401; confidence interval 5 for difference group 1. - group 2:: -5~,6 to 24,8 g), M6G and verapamil were dissmlved in Singer lactate and subsequently m2xed with tylose mucilage, To each rat were administered orally 62,5 g M6G per kg body~weight in tylose mucilage, 15 min before administration of M6G, 10 4 rats received ?0 mg verapamil per kg baby weight orally administered in t;~lose mucilage, For the determination of the pl~~na concentrations of M6G, morphine and M3G, in the case of each rat 6 blbod samples were taken (each about 200 N.l) at the following times: before the administration of M6G, as well as 1.", 2, 4, 6 and 8 hours after M6G administration, The blood samples were transferred into heparinise3 EDTg s~rnthetic resin test tubes and immediately centrifuged, Until analysis, the prepared blood samples were stored at -20oC, The concentration of M6G, morphine and morphine-3-glucur-onide (M3G) were determined by means of HPI~C (cf, Hartley R ". Biomed, Chromatog, (1993) ?:. 34-3?). The detection limit lay for all three substances at 10 ng/ml, i,e, 35.05 nmol/1 for morphine and 22,45 nmol/1 for the morphine glucuronides, In the whole calibration range, the variation coefficient (10 - 500 ng/ml) lay below ll~,.
Inhibition of microbial beta-glucuronidase b~ verapamil From Example 5 is to be seen that a cleavage of glucuronides (M6G) takes place in the intestines of the rat, It is not to be seen whether beta=glucuronidases of the rat and/o_r microbial beta-glucuro~iidases (e, g. E,. coli) a-re responsible for this cleavage, In order to clarify this guestion, beta-glucuronidases from rat intestine homogenates and from E, coli were incubated with verapamil or D-glucarict acid-1,4-lactone in the presence of 4-methylumbellifer~l-~-D-glucuronide AMENDED
SHEET
11_ (MUG), The cleavage of the 4-methylumbelliferyl-~-D-glucuronide is a measure for the activity of the beta-glucuronidase, As is to be expected, D-glucaric acid-1,4-lactone inhibits not only the beta-glucuronidase activity of the rat intestine homogenates but also the E, coli beta-glucuronidase (Fig, 6A and B), Surprisingly, the bactern;al enzyme was clearly inhibited by verapamil (IC50 = 30 ~u.M), whereas the iet beta-glucuronidase is not measurably influenced by verapamil (E-2g, 6A and B), The carrying out of the experiment is described in Example 6, Example 6 Inhibition of 4-methylumbellifervl-~-D-glucuronide (MUG) cleavage by verapamil and D-glucaric acid-1,4-lactpne (A rat, B E, coli)(Fig,. 6), Deep frozen tissue powder of a rat mucosa (duodenum and jej unum) was suspended in 20 mrI Tris-HG~1, phi '7,.~, 1 mM EDTA, 1 mM pefabloc ~ (firm Roth, Karlsruhe, Germany), The protein concentration was determined according to the method of Lowry_ ~~owry a? H,, J, Biol, them, (1951) 193: 265-27~~, The incubation and analysis took place according to:~ L~'perker B, , J,. Pharmacol, Exp,.
Ther, (1997) 28 1:: 914-9207, 50 ~.1 incubation mixture c ontained 2,25 ~g rat protein homogenate or 110 pg (0,001 units) purified E, coli beta-glucuronidase (firm Sigma, Deisenhofen, Germany), The test buffer contained 0,2 mM MUG (firm Sigma, Deisenhofen, Germany),, The incubation mixtures were mixed at 37°C with verapamil or D-glucaric acid-1,4-lactone, After 10 minutes, the MUG buffer was added, After 1 hour at 3~oC, the enzymatic reaction was stopped by addition of 150 ~1 200 mM s odium carbonate solution, After centrifuging (5 min,, 13,000 r,p,m,), the supernatants were analysed by means of HPZC (fluorescence: absorption 355 nm, emission 460 nm), The enzyme activity was correlated __ with the liberation of 4-methylumbelliferone (MU), AMENDED
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The experiments were carried out at the corresponding optima of the beta-glucuronidases (pH '7,0 E, coli or pH 5,O rat), The results of Fig,. 6 show that verapamil is not able to inhibit the glucuronidase of the rat but is a good inhibitor for the bacterial glucuronidase from E, coli, On the other hand, the known inhibitor D-glucaric acid 1,4-lactone inhibits both enzymes equally well AMENDED
The glucuronic acid conjugates can, howeverv, also be cleaved by catalytic action of glucuronidases:into glucuronic acid and in to the starting product,. The cleavage of glucuronides frequently takes place after excretion thereof via the bile in deeper lying small intestine sections or in the large intestine, The thereby resulting starting substances can again be resorbed~snd this become renewed a cttEve in the organism. This process, designated as enterohepatic circulation, can prolong the desired action of substances but can also increase the toxic actions of poisonous substances.
' CA 02376199 2001-12-04 By medicamentous regulation of the beta-glucuronidase activity in the various tissues, new therapy concepts are opened up,.
Use of glucuronidase inhibitors its cancer therapy, A peculiarity of cancer tissues is their high con-centration of bets-glucuronidaees or an extremely high glucuronidase activity, Closely associated with the increased glucuronidase activity is the tendency to form certain tumour meta~~tases, By general administration of a beta-glucuronidase inhibitor, in the case of tumours which, on the basis of the increased beta-glucuronidase ac-tivity, tend to the progression and metastasis formation, the tumour spreading out is seduced via the inhibition of the tumour glucuronidase, Saccharo-1,4-lactose, 2-acetamidoglycal and heparin derivatives were tested for :this purpose /Bernacki R,J " Cancer Metastasis Rev,, (1985) 4: 81 - lOl;Nakajima M,, Journal of Cellular Biochemistry (1988) 36: 15~ - 16'7; Niwa T,, Journal of Biochemistry (1972 ) '72: 20'7 - 211, In most recent times, selective glucuronidase inhibitors have been s9nthe~d (Bosslet K~, EP 0822192), Besides the general use for the therapy, glucuronid~rse .
inhibitors can also be used supportingly in the chemo-therapy of cancer patients f or the increasing of the desired effect in the case of simultaneous reduction of the undesired actions,.
The chemotherapy causes an extraordinary physical and psychic stressing of the cancer patient, Giucuronidase inhibitors can ameliorate negative actions of the chemo-therapy and simultaneously increase the effectiveness of the therapy, For this purpose, the following starting points present themselves, Chemotherapeutics are, inter alia, also excreted via their glucuronides via small intestines, Due to the actions of the there-present glucuronidases, there takes place a cleavage of these glucuronides and liberation ~
of the active cell-toxic substances which damage the intestinal tissue present in continuous cell division and regeneration, For the patient, there result therefrom nausea, vomiting and diarrhoea, combined with a fluid and weight loss, Beta-glucuronidase inhibitors can protect the intest-ines against toxic products from cytostatic gluc~uronides, Thus,e,g, the intestinal toxicity of the anti-tumour agent irinote.ctan hydrochloride can be minimised by preventative 1!Q administration of the beta-glucuronidase inhibitor baicalin, The patients arethe~s protected against a massive diarrhoea and the fhuid losses involved therewith L~akasuna K, Jpn,.
Cancer Res, (1995) 86:: 9'78 - 84;: Kemataki T, HB~, Pat, 5,44,719).
Consideraaions exist of using the cleavage of glucur-onide in certain tissues in order to liberate the active substances from inactive: precursors of a~:ctive medicament (prodrugs), Due to the preferred. liberation in the dises ed target tissues, via the inc:ressed substance concentration, there can be achieved a more or less local action in the case of low ~r~stemic actia~n ~~p~erker B,.,, Clin,. Phsrmakinet, (1_997) 33:: 18 - 31~,. This therapy possibility would be of interest above all in the case of the use of s~.de effect-rich substances in tumour therapy 5 bs9cause tHe. d.e irked aytaaogic properties of chemothera-peutics can be concentrated on the tumour tissues" The tumour progression and the me sstasis formaaion is frequently bound up with an inc:rea ~ed gl.ucuronida~se activity,. In necrotic tumour regions, sn ~~ncraa:aed ~0 glucuronidase activity is present in the extrscel7~uTar space wherees in the healthy tissue the glucuronida a activity is substantially intracellular localised, A
pH value in the tumour displaced towards acid can again increase the activity of the beta-glucuronidase, These 35 physiological conditions offer starting points for the application of glucuronic acid conjugates with c hemotherapeutics to tumour patients for the local ' ~ CA 02376199 2001-12-04 .~5_ liberation of the active substrate after cleavage b9 the:
locally increased glucuronidase activity LSperrker B,.,.
Clin, Pharmacokinet, (199?) 33:~ 18 - 317, The local action could be strengthenedby simultaneous administration of a glucuronide prodrug and of, s tumour-specific antibody which is covalently bound with beta-glucuronidase (antibody-directed prodrug therapy = ADEPT)LSperker B,, CZin, Pharmacokinet, (1997) . 33:~ 18 - 317, The increased tumour selectivity of glucuronide prodrugs leads iro correspondingly higher active material levels in the tumour and sumultaneously to lower native material concentrations in healthy tissue regions, i,e, the effectivenesse~ and compatabilities of the chemo-therapeutics are increased, Known examples are doxorubicin glucuronide prodrugs which, in comparison with the free doxorubicin, make .possible in tumour tissues an about 10 times higher doxorubicin level but, at the same time, protects healthy tissue with a lower concentration so that e,g, the typical cardiotoxic property ~f doxorubicin only plays a subsidiary role L$osslet K,, Cell Biophys, (1994) 24-25;:
51-63; Bosslet K,, Cancer Res, (1994) ~.: 2151-g;
Bosslet K,, Cancer Res, (1998): 1195 - 201; Murdter, T,E "
Cancer Res, (199'7) 5~: 2440-~~, None of these investigations has hitherto lead to therapeutically usable results, i,e, utilisable medicaments, Description of the invention The invention has set itself the task of finding glucuronidase inhibitors which are otherwise pharma-cologically not or only little effective, i,e, display few side reactions, in order to use these as medicaments in the above-described uses alone or in combination with other medicaments for the increasing of the therapeutic breadth, New pales 6 - 11 filed 15th December, 2000 In the case of administration of biologically-inactive glucuronide prodcugs, together with a beta-glucuronidase inhibitor, the cleavage into the effective substrate is delayed so that, in the case of prodrugs with long elimination half value time, the systemic availability - is prolonged,. Correspondingly, the dose can be reduced end the dose ging interval lengthened, Im the case of the tumour-specific prodaug therapy, by additional administration of a cell membrane-premeable IO beta-glucuronidase inhibitor, such as verapamil, the therapeutic breadth is thereby increased that the sub-star~t~.slly intracellularly present beta-glucuronida se is inhibited in healthy tissue and a pharmacological action is~thereby hindered, In the tumour tissue, due to the physiological or due to the glucuronidase concentration increased by ADEPT therapy, the effective substrate is, as previously, formed in the case of suitable choice of dose,.
The inhibiting action on the beta-glucuronidase activity claimed in the invention is verified in the results set out in the following,.
Iimestigations of the lowering of human ~-glucuronidase activity by verapamil, its metabolites and gallopamilr The calcium antagonist verapamil (not only racemate but also both enantiomers), its metabolites and the derivative gallopamil aae in the position Via' Lower the activity of the human ~-glucuronidases, A direct inhibition of the ~-glucuronidase sctivity could be shown in experiments with human ILiver homogenates, For this purpose, homogenates of various liver samples were: incubated with 2,.5 mM 4-methyl-belliferyl-~-D.t.
glucuronide (MUG) and analysed by means of Hi'~C, The:
concentrations of the liberated 4-methylumbelliferone is a measure of the activity of the ~-glucaronidase, In the case of homo mates which, in addition to MUG, also received T00 ~-M verapamil ( racemat ~), the activity was reduced significantly by about 25~, in comps~ison with AMENDED
SHEET
the control samples (Fig, 1).
Parallel bring about verapamil, the metabolite nor-verapamil, D~02, D~03 and gallopamil in the human hepatoma cell Mine HepG2 after 48 h incubation a reduction of the ~-glucuronidase activity to 50 - 65~ which is to be attributed to a reduced expression of the enzyme,.
This reduction of the activity in concentration dependent (Fig. 2).
The reduction of the ~-glucuronidase a3ctivity carz~ld be observed eqw-311y strongly with verapamil racemate and with R- and S-verapamil, fihe metabolites no~verapamil, D'702 and D703 show a comparable influence on the activity of the ~-glucuronidase in HepG2 cells, The incubation with D61~, a further metabolitew, only brings about a lowering of the activity by' 12~ whicli, however, ~s not statistically significant, Gallopamil brings about an effect comparable to verapamil (Fig" 3), -Example 1 Inhibition of the activity of human liver ~-glucuronidase by verapamil (F.2g, 1), Human liver homogenates were incubated with the enzyme substrate 4-methylbelliferyl-~-D-glucuronide (1 h, 3'7°C), 100 ~.M verapamil or DMSO (control) were added to the reaction mixture, The liberation of 4-methylumbelli-ferone was measured by mearns of HPI~C analysis ('~ significant difference to the control; p ~ 0001; n =
3 independent experiments), Exa mp 1 a 2 Concentration dependency of the verapamil action in the human hepatoma cell Mine HepG2 (Fig, 2), HepG2 cells were incubated for 48 h at 3'7oC with the concentrations of verapamil given in Fig, 2, A~'ter ~.~rsie of the cells, in each case 2,25 ~.g of cellular protein were incubated (2 h, 3'7°C) with ~-glucuronidase substrate 4-methylumbelliferyl-~-D-glucuronide and the concentration of the liberated 4-methylumbelliferone measured by means of HPI~C ( '* significant difference ~o the control, p <0~05), HMENDED
SHEET
Example 3 Lowering of the ~-glucuronidase activity in HepG2 cells by incubation with verapamil, verapsmil metabolites sand gallopamil (Fig, 3), HepG2 cells were incubated for 48 h,~t 37°C with 100 ~M verapamil (Yera), in each ceae 100 rM DFl'7, D~02, D'703, 30 ~,.M norverapamil (Nor) or 100 ~M gallopamil (Gallo), After lysis of the cells, the ~-glucuronidaee activity was determined by means of 4-methylumbelliferyl-~i-D-glucuronide cleavage (significant difference to the control, '~ P < 0,01, *~ g ~ 0,.001, n = 3 independent -experiments), Example 4 Lowering of the beta-glucuronidase expression by vera~amil - 15 in the human hepatoma cell line HepG2 (Fig,. 4), HepG2 cells were- incubsrted 48 h at 3~°C with 100 ~M
verapamiT or DMSO (control), After lysis of the cells, 50 ~g cellular protein were separated off by means of SDS page,. transferred to nitrocellulose and subsequently incubated with the monoclonal antibody 2156/42, The band intensity was determined densitometrically (DE = densito-metric units; 'significant difference to the control, p ~ 0,05; n = 3 independent experiments), Inhibition of the glucuronidases in the rat intestine by verapamil (comparison) In a study with Sprague-Dawley rats, the absorption of orally administered morphine-6-glucuronide (M6G) to twa groups .~. - (group- 1~. 5, without verapamil administ-ration; group 2: n = 4 previous verapamil afministration) was investigated, The study was carried out with rats since these cannot form M6G metabolically from morphine (Aasmundstad T,A,, Biochem, P.harmacol, (1993) 46:
961-968) so that the M6G measured in the plasma origi.na.r~ed= exclusively from the absorption of the orally administered M6G, Whereas the previous administration of verspamil had no influence on the height of the plasma concentration AMENDED
SHEET
of M6G or its variation in time, the concentrations of morphine and M3G in the case of previous verapamil administration (group 2) were distinctly smaller than in the case of the group without verapamil (group 1) (Fig. 5).
The absent influence on the height of the plasma concentration of M6G or its variation in time makes it improbable that the reduction of the morphine and M3G
absorption depends upon an inhmbition of the intestinal mobility L~hah M~H" J, Pharm, Pharmacol, (198?) 39:
103?-1038; Krevsky B,, Dig,. Dis.. Sci, (1992) 37:
919-9247, It is known that M6G inhibits the intestinal motility with the same potency as morphine ~Schmidt N,, Eur, J, Pharmacol, (1994) 255 245-237, An increase of this inhibition by verapamil ,L~hah N~,.H,., J,. Pharm, Pharmacol,. (198?) 39, 1037-103_87 acts with all probability on M6G and morphine to the same extent, On t he other hand, only the plasma level of morphine Qr M3G but not of M6G were reduced, i,e, the cleavage of M6G available after oral administration to morphine is thus inhibited, Therefrom result lower morphine and, as a result, M3G
plasma levels since the greater part of the absorbed morphine i5 metabolised by glucurony~. transfera.ses to M3G, The carrying out of the experiments i~ described in Example 5, Example 5 Plasma concentration time progression of morphine-6-glucuronide (M6G), morphine and morphine-3-glucuronide (M3G) after oral administration to Sprague-Ds~wley rats of M6G with or without pervious oral administration of verapamil (Fig,. 5), The investzigation was carried out on 9 male S:prsgue-Dawley rats, The rats were divided into 2 groups:: group T
(5 animals, weight: 258 + 31.2 g) received only 62,5 mg/kg morphine-6-glucuronide (M6G) administered orally, Group,2 (4 animals, weight 2?2 +-8 g) received, 15 minutes AMENDED
SHEET
before M6G administration (62,5 mg/kg orally-, ?0 mg/kg verapamil orally administered, The groups dia not differ significantly from ane another with regard to their weight (t-test: t = -0,923, p = 0,401; confidence interval 5 for difference group 1. - group 2:: -5~,6 to 24,8 g), M6G and verapamil were dissmlved in Singer lactate and subsequently m2xed with tylose mucilage, To each rat were administered orally 62,5 g M6G per kg body~weight in tylose mucilage, 15 min before administration of M6G, 10 4 rats received ?0 mg verapamil per kg baby weight orally administered in t;~lose mucilage, For the determination of the pl~~na concentrations of M6G, morphine and M3G, in the case of each rat 6 blbod samples were taken (each about 200 N.l) at the following times: before the administration of M6G, as well as 1.", 2, 4, 6 and 8 hours after M6G administration, The blood samples were transferred into heparinise3 EDTg s~rnthetic resin test tubes and immediately centrifuged, Until analysis, the prepared blood samples were stored at -20oC, The concentration of M6G, morphine and morphine-3-glucur-onide (M3G) were determined by means of HPI~C (cf, Hartley R ". Biomed, Chromatog, (1993) ?:. 34-3?). The detection limit lay for all three substances at 10 ng/ml, i,e, 35.05 nmol/1 for morphine and 22,45 nmol/1 for the morphine glucuronides, In the whole calibration range, the variation coefficient (10 - 500 ng/ml) lay below ll~,.
Inhibition of microbial beta-glucuronidase b~ verapamil From Example 5 is to be seen that a cleavage of glucuronides (M6G) takes place in the intestines of the rat, It is not to be seen whether beta=glucuronidases of the rat and/o_r microbial beta-glucuro~iidases (e, g. E,. coli) a-re responsible for this cleavage, In order to clarify this guestion, beta-glucuronidases from rat intestine homogenates and from E, coli were incubated with verapamil or D-glucarict acid-1,4-lactone in the presence of 4-methylumbellifer~l-~-D-glucuronide AMENDED
SHEET
11_ (MUG), The cleavage of the 4-methylumbelliferyl-~-D-glucuronide is a measure for the activity of the beta-glucuronidase, As is to be expected, D-glucaric acid-1,4-lactone inhibits not only the beta-glucuronidase activity of the rat intestine homogenates but also the E, coli beta-glucuronidase (Fig, 6A and B), Surprisingly, the bactern;al enzyme was clearly inhibited by verapamil (IC50 = 30 ~u.M), whereas the iet beta-glucuronidase is not measurably influenced by verapamil (E-2g, 6A and B), The carrying out of the experiment is described in Example 6, Example 6 Inhibition of 4-methylumbellifervl-~-D-glucuronide (MUG) cleavage by verapamil and D-glucaric acid-1,4-lactpne (A rat, B E, coli)(Fig,. 6), Deep frozen tissue powder of a rat mucosa (duodenum and jej unum) was suspended in 20 mrI Tris-HG~1, phi '7,.~, 1 mM EDTA, 1 mM pefabloc ~ (firm Roth, Karlsruhe, Germany), The protein concentration was determined according to the method of Lowry_ ~~owry a? H,, J, Biol, them, (1951) 193: 265-27~~, The incubation and analysis took place according to:~ L~'perker B, , J,. Pharmacol, Exp,.
Ther, (1997) 28 1:: 914-9207, 50 ~.1 incubation mixture c ontained 2,25 ~g rat protein homogenate or 110 pg (0,001 units) purified E, coli beta-glucuronidase (firm Sigma, Deisenhofen, Germany), The test buffer contained 0,2 mM MUG (firm Sigma, Deisenhofen, Germany),, The incubation mixtures were mixed at 37°C with verapamil or D-glucaric acid-1,4-lactone, After 10 minutes, the MUG buffer was added, After 1 hour at 3~oC, the enzymatic reaction was stopped by addition of 150 ~1 200 mM s odium carbonate solution, After centrifuging (5 min,, 13,000 r,p,m,), the supernatants were analysed by means of HPZC (fluorescence: absorption 355 nm, emission 460 nm), The enzyme activity was correlated __ with the liberation of 4-methylumbelliferone (MU), AMENDED
SHEET
The experiments were carried out at the corresponding optima of the beta-glucuronidases (pH '7,0 E, coli or pH 5,O rat), The results of Fig,. 6 show that verapamil is not able to inhibit the glucuronidase of the rat but is a good inhibitor for the bacterial glucuronidase from E, coli, On the other hand, the known inhibitor D-glucaric acid 1,4-lactone inhibits both enzymes equally well AMENDED
Claims (6)
1. Use of R-verapamil or R-gallopamil or their salts with pharmaceutically compatible acids for the preparation of medicaments for the stabilisation of glucuronide-active material conjugates against human tissue glucuronidase in human blood or, intestinal tract.
2. Use according to claim 1, characterised in that the R-enantiomers are used in pure form or, in comparison with the racemate, in enriched form.
3. Use according to claim 1 or 2, characterised in that the glucuronidase inhibitor is used, with suitable pharmacologically compatible adjuvants, for the preparation of orally or parenterally normally liberating or controlled liberating medicaments.
4, Use according to one or claims 1 to 3, characterised in that the glucuronidase inhibitor is used for the stabilisation of metabolically-formed glucuronide conjugates of side effect-rich active materials in order to reduce their side effects or to introduced a detoxification.
5. Use according to one of claims 1 to 3, characterised in that the glucuronidase inhibitor is used combined with a glucuronide conjugate o~ an inflammation-inhibiting active material to be taken orally in order to protect this in the upper stomach-intestine tract against a cleavage and resorption and to activate in the deeper lying intestinal sections by cleavage for the intestinal local therapy.
6. Use according to one of claims 1 to 3 for the improvement of the tissue-specific therapy, characterised in that the glucuronidase inhibitor, in the case of combined use with a glucuronide prodrug, protects this against activation in healthy tissue in the case of maintenance of the activation in the target tissue.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19925810A DE19925810A1 (en) | 1999-06-07 | 1999-06-07 | Use of verapamil and verapamil derivatives for the manufacture of medicinal products with glucuronidase inhibitory activity |
| DE19925810.4 | 1999-06-07 | ||
| PCT/EP2000/004848 WO2000074670A1 (en) | 1999-06-07 | 2000-05-27 | USE OF VERAPAMIL AND VERAPAMIL DERIVATIVES FOR PRODUCING MEDICAMENTS WITH AN INHIBITING EFFECT ON β-GLUCURONIDASE IN HUMAN TISSUE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2376199A1 true CA2376199A1 (en) | 2000-12-14 |
Family
ID=7910362
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002376199A Abandoned CA2376199A1 (en) | 1999-06-07 | 2000-05-27 | Use of verapamil and verapamil derivatives for producing medicaments with an inhibiting effect on .beta.-glucuronidase in human tissue |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1183023A1 (en) |
| JP (1) | JP2003501384A (en) |
| CA (1) | CA2376199A1 (en) |
| DE (1) | DE19925810A1 (en) |
| WO (1) | WO2000074670A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10106530C2 (en) * | 2001-02-13 | 2002-06-27 | Heinz Kiefer | Medicines to detoxify the body |
| CN101802189B (en) * | 2007-09-20 | 2013-03-27 | 花王株式会社 | Beta-glucuronidase inhibitor |
| RU2532027C2 (en) | 2009-07-24 | 2014-10-27 | Мика Фарма Гезельшафт Фюр Ди Энтвиклюнг Унд Фермарктунг Фармацойтишер Продукте Мбх | Foam-forming liquid compositions containing active agents, and methods for preparing and developing thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0753665B2 (en) * | 1984-04-20 | 1995-06-07 | 財団法人癌研究会 | Anti-metastatic agent |
| DE3635930A1 (en) * | 1986-10-22 | 1988-04-28 | Basf Ag | ACTIVE SUBSTANCES FOR TUMOR TREATMENT |
| JPH0798752B2 (en) * | 1991-08-09 | 1995-10-25 | 株式会社ツムラ | β-glucuronidase inhibitor |
| DE4236237A1 (en) * | 1992-10-27 | 1994-04-28 | Behringwerke Ag | Prodrugs, their preparation and use as medicines |
| EP0647450A1 (en) * | 1993-09-09 | 1995-04-12 | BEHRINGWERKE Aktiengesellschaft | Improved prodrugs for enzyme mediated activation |
| DE19631288A1 (en) * | 1996-08-02 | 1998-02-05 | Hoechst Ag | New inhibitors of β-glucuronidase |
-
1999
- 1999-06-07 DE DE19925810A patent/DE19925810A1/en not_active Withdrawn
-
2000
- 2000-05-27 EP EP00931265A patent/EP1183023A1/en not_active Withdrawn
- 2000-05-27 WO PCT/EP2000/004848 patent/WO2000074670A1/en not_active Application Discontinuation
- 2000-05-27 CA CA002376199A patent/CA2376199A1/en not_active Abandoned
- 2000-05-27 JP JP2001501207A patent/JP2003501384A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000074670A1 (en) | 2000-12-14 |
| DE19925810A1 (en) | 2000-12-14 |
| JP2003501384A (en) | 2003-01-14 |
| EP1183023A1 (en) | 2002-03-06 |
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